Porphyromonas gingivalis is a periodontal pathogen implicated in the initiation and progression of chronic periodontitis in adults. We recently demonstrated that this organism produces two novel classes of serine lipids that inhibit bone cell function and activate macrophages to release important cytokines. At least one of these serine lipids also mediates its effects on bone cells and macrophages through engagement of the innate immune system, specifically through Toll Receptor 2 (TLR2). Understanding how these lipids promote TLR2- dependent cellular effects is relevant specifically to the reported effects of P. gingivalis on periodontal bone loss in experimental animals. Since all Bacteroidetes appear to be capable of producing these serine lipids, including intestinal Bacteroidetes, their role in other medical diseases, such as atherosclerosis, may also be important. P. gingivalis serine lipids are unusual in that a mammalian enzyme called phospholipase A2 (PLA2) can hydrolyze the dominant serine lipid to a de-esterified form that is even more potent than the parent lipid in inhibiting bone cells. Of note, chronic inflammation is associated with increased expression of PLA2 which results in elevated prostaglandin levels within chronically inflamed tissues. However, only one chiral form of the parent serine lipid is hydrolyzed by PLA2. This application proposes to first quantify the levels of each chiral form of the parent serine lipids in common oral Bacteroidetes. Next, we will investigate hydrolysis of the each chiral form of the parent serine lipid by macrophages and osteoblasts. This will determine the importance of intracellular versus extracellular hydrolysis of the different chiral forms and the relevance of the location of lipid hydrolysis to biological activity. Finally, we will determine which cellular PLA2 enzyme classes are responsible for the hydrolysis of the parent lipid. We will examine the role of TLR2 in these processes by comparing bacterial lipid effects in bone cells isolated from either wild type or TLR2 knockout animals. The experiments summarized in this proposal are critical to explaining how P. gingivalis promotes TLR2-dependent bone loss in periodontal diseases. In addition, the discovery that the PLA2 enzyme will hydrolyze the parent serine lipid to a de-esterified serine lipid of greater potency is potentially important in other medical conditions where bacterial lipids accumulate, including atherosclerotic plaques.